The goal of this project is to determine the mechanism(s) and physiological impact of endogenous superoxide production in Escherichia coli. Strains that lack superoxide dismutase exhibit severe aerobic growth deficiencies, indicating that superoxide is made intracellularly during aerobiosis. Our studies have indicated that superoxide is formed primarily by autoxidation of components of the respiratory chain, including fumarate reductase. We now propose: (1) to identify the other sites of respiratory superoxide production; (2) to determine how the electronic structure of fumarate reductase singularly predisposes it to generate superoxide; (3) to establish whether the superoxide flux from respiratory sources is large enough to damage SOD-proficient cells; and (4) to determine whether superoxide production by low-potential anaerobic respiratory chains causes acute oxidative stress when this facultative anaerobe enters an aerobic environment. For years workers have imposed artificial stresses upon E. coli in order to characterize its elaborate network of oxidative defenses. We want to identify the endogenous sources of the oxidants against which E. coli has evolved to defend itself. More generally, this investigation will identify the characteristics that predispose electron carriers to generate superoxide. Such information may assist in identifying similar sources of superoxide in mammalian cells. Recent work indicates that endogenous superoxide causes the familial form of the neurological disorder amyotrophic lateral sclerosis. Circumstantial evidence has suggested that superoxide may also have a role in the initiation or progression of other clinical disorders, including heart disease and cancer.